JP2001521174A - Methods for detecting abused drugs in biological samples - Google Patents

Abstract

(57) Abstract: A high-purity styrene-divinylbenzene resin is used for rapid solid-phase extraction of an abused drug from a body fluid sample. A simple extraction method is described, which provides high recovery and selectivity for a variety of drugs. A convenient method for increasing the flow rate in such solid phase extraction is also described.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION

The present invention relates to a method for rapidly solid-phase extraction of various abused drugs from a body fluid sample. The method provides a simple extraction method for a variety of drugs using a single adsorbent and achieves high recovery.

[0002]

[References]

 Chen, XH et al., J. Anal.Toxicol. 16: 351 (1992) Goldberger, BA et al., J. Chromatogr.A 674 (1-2): 73-86 (1994) Logan, BK et al. , J. Anal. Toxicol. 14: 154 (1990)

[0003]

BACKGROUND OF THE INVENTION

Detection and identification of harmful or regulatory substances in body fluid or tissue samples is an important part of clinical and forensic medicine. This process is often complicated by the large number of endogenous components present in a typical biological sample from which the target substance must be isolated. It is desirable that such identification be performed quickly with minimal sample preparation and extraction steps. A high recovery rate is required for the detection of trace amounts and, if necessary, for accuracy in quantification.

[0004] Solid phase extraction (SPE) is more convenient and has largely replaced liquid-liquid extraction due to the higher recovery of commonly noted substances. Since the same equipment, materials and methods are preferably used for the maximum number of different substances to be identified,
A "mixed" extraction medium containing both hydrophobic and polar substances has been introduced. These allow a variety of interactions with various parts of the drug molecule, ie, both the carbohydrate domain and the polar or charged sites. For cation and anion exchange resins, different resins are used for differently charged analytes.

[0005] Typically, however, the use of such a mixed support selectively binds the compound of interest, elutes contaminants, and then selectively elutes the target compound, A very complex extraction procedure using a series of extraction steps and / or complex solvent mixtures was required. Even with such methods, analyte recovery may be low and interfering endogenous substances may still elute at the same time. In addition, contamination from the support itself can also prevent identification.

[0006] It is therefore desirable to provide a single support and a convenient extraction method that are suitable for a wide range of substances to be analyzed, especially for detecting abused drugs in body fluid samples.
Ideally, extraction without further purification is appropriate for gas chromatography (GC), GC-MS (mass spectrometry), or HPLC (high-performance liquid chromatography) analysis, which allows the High turnover can be achieved while minimizing contamination.

[0007]

Summary of the Invention

In one aspect, the invention provides a diagnostic method for determining the presence and / or amount of an abused drug or a metabolite or derivative thereof in an aqueous body fluid sample. According to this method, a bodily fluid sample, preferably a urine sample, suspected of containing an abused drug or an analyte that is a metabolite or derivative thereof, is contacted with a substantially underivatized styrene-divinylbenzene resin adsorbent. Then, the analyte is adsorbed on the adsorbent. The sorbent is then washed with a first solvent or solvent system effective to selectively remove non-analyte components from the sorbent while the analyte remains bound to the sorbent. The adsorbent is then flushed with a second solvent or solvent system that is effective to selectively elute the analyte from the adsorbent.

[0008] Thereafter, the analyte in the eluate is detected and / or quantified. Suitable methods of detection or quantification are gas chromatography (GC), GC-mass spectrometry (MS), GC-MS-MS or HPLC (high performance liquid chromatography). Generally, this method results in an eluate that contains about 80% or more, preferably 90% or more, of the analyte initially present in the sample and that does not need to be further purified for gas chromatography analysis. In a preferred embodiment of the method, the abused drug or metabolite is THC carboxylic acid, heroin, morphine, 6-acetylmorphine, morphine-3-glucuronide, codeine, cocaine, benzoylecgonine, ecgonine methyl ester, amphetamine, It is selected from the group consisting of methamphetamine, lysergic acid diethylamide (LSD), phencyclidine (PCP), barbiturate, benzodiazepine or a metabolite thereof, and a tricyclic antidepressant or a metabolite thereof. Barbiturates include, for example, butabarbital, pentobarbital, secobarbital, phenobarbital, alfenal or amobarbital. Benzodiazepines and metabolites for which the method is useful include oxyazepane, chlorodiazepoxide, hydroxyalprazolam, and hydroxytriazolam. The tricyclic antidepressant (TCA) may be, for example, doxepin, imipramine, or amitriptyline, and the TCA metabolite may be, for example, nordoxepin, desipramine, or nortriptyline.

[0009] The styrene-divinylbenzene adsorbent used in the extraction includes a high-purity resin such that extraction of about 1.0 g of the resin with 10 ml of hexane and then 10 ml of methanol results in less than about 0.2 mg of residue. It is preferred that In a preferred method, less than about 100 mg of resin is used for the extraction of a 2 ml urine sample. A preferred solvent combination is that the first (washing) solvent is water or 5% aqueous methanol and the second (eluting) solvent is ethyl acetate. Another one
In one preferred solvent combination, the first solvent system is a weakly acidic aqueous buffer containing about 10% by volume acetonitrile and the second solvent system is about a 1: 1 volume ratio of acetone / fluoroform in acetone / fluoroform. There is something there.

When the analyte is THC-COOH, another preferred solvent combination is an aqueous solution in which the first solvent contains about 5% by volume acetonitrile and 1% by volume 30% aqueous ammonium hydroxide. There is acetonitrile wherein the second solvent system is ethyl acetate / isopropyl alcohol in a volume ratio of about 85:15. When the analyte is an opium such as heroin, morphine, 6-acetylmorphine or codeine or a metabolite or derivative thereof, another preferred solvent combination is where the first solvent is a weakly acidic aqueous buffer and In some cases, the second solvent is methanol containing about 2% by volume of a 30% aqueous ammonium hydroxide solution.

The analyte is an opium such as heroin, morphine, 6-acetylmorphine, morphine-3-glucuronide or codeine, or a metabolite or derivative thereof, and the sample is hydrolyzed before being applied to the sorbent. When used, another preferred solvent combination is that the first solvent is aqueous methanol containing about 7% by volume of methanol in water and the second solvent system is about 1% by volume of 30% hydroxide. There is an approximately 1: 1 volume ratio of n-butyl chloride / ethyl acetate containing an ammonium solution.

[0012] When the analyte is cocaine or benzoylecgonine, another preferred combination of solvents is where the first solvent is a weakly acidic aqueous buffer and the second solvent system is an approximately 1: 1 solution. Some are acetone / chloroform in a quantitative ratio or acetonitrile / n-butyl chloride in a volume ratio of about 3: 7. When the analyte is amphetamine or methamphetamine, another preferred solvent combination is that the first solvent is aqueous methanol containing 5% methanol by volume in water and the second solvent system is methanol There is something.

When the analyte is a barbiturate such as butabarbital, pentobarbital, secobarbital, phenobarbital or amobarbital, another preferred combination of solvents is that the first solvent is a weakly acidic aqueous buffer; In some, the second solvent system is hexane / ethyl acetate in a volume ratio of about 3: 1 or acetone / chloroform in a volume ratio of about 1: 1. When the analyte is PCP, another preferred solvent combination is that the first solvent is a weakly acidic aqueous buffer and the second solvent system is about a 1: 1 volume ratio of acetone / chloroform or acetone / chloroform. Some are methylene chloride.

When the analyte is a benzodiazepine or a metabolite such as benzodiazepine, oxazepam, chlorodiazepoxide, hydroxyalprazolam or hydroxytriazolam, another preferred solvent combination is that the first solvent is a weakly acidic aqueous solution. There is a buffer wherein the second solvent system is acetone / chloroform in a volume ratio of about 1: 1. When the analyte is LSD, another preferred solvent combination is a first solvent is a weakly acidic aqueous buffer containing about 10% by volume acetonitrile and a second solvent system is about 1: 1. There is a volume ratio of acetone / chloroform.

When the analyte is a tricyclic antidepressant or metabolite such as doxepin, imipramine, amitriptyline, nordexepin, desipramine or nortriptyline, another preferred solvent combination is that the first solvent is a weakly acidic one. There is an aqueous buffer or 5% aqueous methanol, wherein the second solvent system is methylene chloride / isopropyl alcohol in a volume ratio of about 4: 1. In another aspect, the present invention provides a method for increasing the flow rate of liquid through a column containing an adsorbent packed bed, where the column has an outlet connected to the underside of the adsorbent bed, such as solid phase extraction. . According to this method, the lower end of the discharge pipe is a tapered tip, so that the flow of the adsorbent bed and the discharge pipe is promoted by capillary action. In a preferred embodiment of the method, the solvent is an aqueous solvent or an aqueous solution and the sorbent is a silica-based sorbent.

[0016]

[Detailed description]

I. Definitions The following terms have the following meanings unless otherwise indicated. "Abuse drugs" include illicit drugs and prescription drugs that undergo abuse or addiction, such as stimulants, tranquilizers, and antidepressants. Examples include THC (tetrahydrocannabinol), heroin, morphine, codeine, cocaine, amphetamine, methamphetamine, LSD (risergic acid diethylamide), PCP (phencyclidine), butabarbital, pentobarbital, secobarbital, phenobarbital, Barbiturates such as alfenal and amobarbital; benzodiazepines such as oxazepam and chlorodiazepoxide; and tricyclic antidepressants such as doxepin, imipramine and amitriptyline. The method also applies to the usual metabolites and derivatives of these substances as described in Section III below.

“Solvent system” as used herein refers to a single solvent or a mixture of solvents. "Gas chromatography (GC) analysis" refers to GC and GC-MS, GC-MS-MS, GC-FI
Includes GC-based methods such as D. II.Support for Solid Phase ExtractionA.Adsorbent According to the present invention, a body fluid sample suspected of containing an abused drug or its metabolites is treated with substantially underivatized styrene-divinyl benzene (also referred to as cross-linked polystyrene). SDVB) Contact with the adsorbent surface of the resin. "Substantially non-derivatized" means that less than about 2% of the styrene moieties of the polymer have been substituted with groups other than DVB bridging groups. Preferably, the resin is not 100% derivatized. Suitable SDVB resins are available from Mitsubishi Chemical, Rohm and Hass, The Purolite Compan
y and commercially available from suppliers such as Polymer Laboratores, Inc.

[0018] Resins having various particle sizes and properties are available. For this application, particles ranging in size from 5 μm to 1000 μm may be used, but to reduce the problem of `` particulates '' passing through the frit or filter, and to obtain acceptable flow rates, 50 μm Is preferable. A size of 100 to 200 μm is particularly preferred. Particles having a pore size of more than 20 mm and up to 10,000 mm may be used, with a pore size of 100 to 500 mm being preferred.

For optimal results, the resin is washed before use to obtain a high purity adsorbent. In a typical washing method, the resin is immersed in toluene overnight, again immersed in toluene overnight, filtered, immersed in acetone overnight, filtered, and dried in a drier. Alternatively, the resin is soaked in 7: 3 n-butyl chloride / acetonitrile overnight and then washed at least twice with isopropyl alcohol. After such a wash, 1 g of the sorbent sample was added to 10 ml of methanol, then 10 ml of hexane, or 1:
After extraction with 1 n-butyl chloride / acetonitrile (20 ml of solvent per gram of resin), evaporation of the solvent typically leaves less than 0.5 mg, preferably less than 0.2 mg of residue. Further, it is preferred that a 1 g sample be extracted with CH ₂ Cl ₂ to obtain 1 μg of any given component as determined by GC-FID (flame ionization detection) analysis.

The use of this high-purity resin minimizes contamination from the resin during sample identification. Otherwise, such contamination can interfere with the quantification of the analyte extracted from the sample or the identification of the analyte, for example, if the contaminant has a similar GC retention time and / or ionic molecular weight as the analyte. Can be For the drug identification method described herein, GC-MS analysis of the resin extracted as described above did not significantly interfere with the key ions of the analyte.

[0021] Extraction methods using a polystyrene adsorbent that have been reported so far usually include GC.
A purification step, such as preparative thin-layer chromatography of the eluate, has been added before performing the analysis. The resulting eluate is directly converted to a GC-based analysis after the analyte has been converted to the desired derivative because contamination from the adsorbent is minimal and the selectivity of the solvent system used in the method is high. May be used. B. Column Configurations While various types of column materials and configurations can be used, conventional short path chromatography columns, such as shown at 10 in FIG. 1, are suitable for inexpensive and easy packing and elution. Column 12 may be obtained from a syringe barrel. The adsorbent 14 is preferably sandwiched between two porous frits as indicated at 16 and 18. The column and frit are preferably made of a strong, non-swelling plastic, such as polystyrene, polypropylene or Teflon. In particular, for applications requiring high purity, a glass column or a Teflon (registered trademark) frit may be used. Preferably, the column and frit components are manufactured such that they contain only minimal amounts of leachable material, in addition to swelling in the solvent used for extraction, and are washed as necessary.

Since this extraction method utilizes the gravitational flow of the solvent, no pressurizing means is required. However, it is desirable to provide a means for decompressing the column outlet or pressurizing the column inlet to dry the solvent bed and increase the flow rate and sample throughput. To detect an abused drug in a body fluid sample such as a urine sample, a 3 ml column with an inner diameter of approximately 1 cm is typically used for a 2 ml liquid sample. The amount of adsorbent is preferably 50 to 20 mg of resin per 2 ml of sample, more preferably less than 100 mg. This amount has been found to provide high solute recovery without unacceptable flow reduction. If desired, the flow rate may be increased by increasing the column diameter. However, if the diameter is too large, the height of the bed will be very low, making it difficult to obtain a uniform and firm packing.

A column improvement that is particularly useful for increasing the flow rate of columns that use smaller particle size adsorbents or finer frits is shown in FIG. As shown, a tapered tip 20, such as a pipette tip, is added below the outlet tube to facilitate flow by capillary action. For example, 75 mg of silica-based sorbent (Bond Elut
Certify® HF, Varian Sample Preparation Products, Harbor City,
The flow time was measured for the flow of an aqueous sample (2 ml urine + 1.5 ml buffer) through a 3 cc column containing 50 mg of styrene-divinylbenzene adsorbent as described herein. The particle size of the adsorbent in each case was about 150μ and was retained on a 20μ frit. In this experiment, the pipette tip used was about 4.5 cm long, the inner diameter at the top was about 0.4 cm, the inner diameter at the lower outlet was about 0.1 cm, and the volume was about 200 μl . The flow times are shown in Table I.

As the data indicates, the column improvement significantly improved the flow time of aqueous samples, especially for silica-based sorbents. Since the flow of the aqueous sample through the hydrophobic SDVB is already quite fast, column modification is less effective in this case.

[0025]

[Table 1]

III. Sample Preparation As is customary in the analysis of body fluid samples, the sample is generally pre-treated to remove potential interfering components such as proteins or particles, or to provide an optimal extract. Adjust the Ph or ionic strength of the sample or dilute the sample for convenience. In the analysis of opiates, samples are often hydrolyzed, for example by treatment with aqueous acids.

[0027] Such pretreatment methods are known in the art (eg, Goldberger, Chen, Lo.
gan). Blood producing samples and urine are typically diluted in a buffer solution. For blood samples, cells may be fragmented using sonication. Proteins may be precipitated, for example, by the addition of organic solvents, perchloric acid or trichloroacetic acid, or metal ions, but such precipitation can cause analyte degradation by co-precipitation. IV. Analytes Preferred analytes for this method are compounds referred to as drugs of abuse.
This includes so-called "street drugs" or illicit drugs, as well as sedatives, antidepressants, etc. that are available by prescription but are subject to abuse or regular use. Specific classes of drugs that can be analyzed according to this method include: 1. THC (tetrahydrocannabinol) THC carboxylic acid * 2. Cocaine benzoylecgonine * 3. Amphetamine methamphetamine 4. Barbiturates Contains butabarbital pentobarbital secobarbital phenobarbital amobarbital alfenal 5.PCP (phencyclidine) 6.opiates, including codeine heroin morphine MAM (6-acetylmorphine) * morphine-3-glucoronide * 7. Benzodiazepines, including oxazepam chlorodiazepoxide hydroxyalprazolam * hydroxytriazolam * 8. LSD (risergic acid diethylamide) 9. Tricyclic antidepressants (TCAs), including nordoxepin * doxepin desiprami * Nortriptyline * Includes imipramine amitriptyline * Metabolites As shown, also includes various metabolites or derivatives of natural drugs, which are often the primary metabolites detected due to the rapid metabolism of drugs in the body. Become a substance. For example, THC (11-nor- △ -9-tetrahydrocannabinol), a major psychoactive compound in cannabis, rapidly accumulates in tissues of the body because of its lipophilicity and is detected in blood or urine. Difficult to do. However, its major metabolite, THC-carboxylic acid, is secreted in urine as glucuronic acid at detectable levels.

Cocaine is rapidly metabolized in the body to benzoylecgonine, ecgonine methyl ester and other minor metabolites. Benzoylecgonine can be detected in urine for a longer time than ecgonine methyl ester, and therefore its detection is
It is the primary means of screening for cocaine use. The most common opiates include morphine, heroin and codeine.
Morphine is also a major metabolite of codeine. 6-, the main metabolite of heroin
Acetyl morphine (also called monoacetyl morphine, MAM or 6-MAM)
It is formed by deacetylation of heroin (3,6-diacetylmorphine), which occurs quickly after ingestion. MAM is further metabolized to morphine and morphine-3-glucuronide. All three of these products, as well as traces of natural heroin, are found in urine up to 40 hours after heroin administration. V. Extraction Method A. Procedure In accordance with this method, a body fluid sample suspected of containing an abused drug or metabolite, especially one of the analytes listed above, is optionally pre-treated to provide a highly pure substantially derivative. It was applied to a column of unmodified SDVB resin. Upon contact with the resin, the analyte and typically other components of the sample adsorb to the resin. The resin was then washed with a solvent or solvent system effective to selectively remove some or all of the non-analyte components while the analyte remained bound to the resin.

After washing, a second solvent is applied that is effective to selectively elute the analyte from the column. While it is preferred to elute only these components, generally the solvent is selected so that components that interfere with subsequent analyte identification are not eluted to a significant degree. The column washing operation, sample application, and sample elution are preferably performed under gravity flow conditions. A vacuum treatment is often used to dry the column between these operations, and a small vacuum may be used to withdraw the solution from the column.
However, it is not necessary to apply a high vacuum.

Examples 1-10 below describe methods and preferred solvent systems for detecting common abused drugs or their metabolites in urine. Compared to many procedures previously used in the art that require several washing and elution steps (e.g., mixed columns), the procedures described below involve only one washing step and one elution step do not need. Many of these methods use washing with a weakly acidic buffer. The pH of the buffer in these examples preferably ranges from about 4.5 to about 6.5,
A typical pH is 6.0. Although the preferred solvent mixture proportions are given in these examples, in general these proportions can be varied slightly without significantly affecting the recovery or selectivity. In addition, sample elution buffer,
Reducing the volume of column conditioning, wash, or elution solvent did not have a significant effect on the final product recovery or purity in most cases.

B. Analyte Recovery According to a further advantage of the present method, high recovery rates are achieved. The analyte recoveries obtained using the methods described in the examples are shown in Table II. As shown in the table, typically more than 80%, often more than 90%, of the analyte present in the body fluid sample is recovered in the eluate. In some cases, recoveries slightly higher than 100% are seen. This is generally due to the presence of trace contaminants and / or small errors in quantification relative to the internal standard.

While a number of solvent systems were tested, those described in the examples provide recovery as shown in Table I for selected analyte groups and GC-MS and HPLC scans discussed below. It was found to show the best combination of selectivity as shown by. Certain solvent systems, such as those described in Examples 8 and 10, have shown good results for a variety of analytes.

[0033]

[Table 2]

In contrast to these results, the widely used poly (methyl methacrylate) (PMMA)
Commercial extraction systems using base-based sorbents are available from the manufacturer (Biochemical Diagnostics
, Inc., Edgewood, NY), the following recoveries were obtained using the extraction protocol. The extraction protocol tested both the original (1995) and the revised (1997). Original Revision THC-COOH 50% 60% Cocaine 87% 10% Benzoylecgonine 14% 90% Codeine 100% 66% Morphine 26% 44% 6-MAM 111% 39% As can be seen, about THC-COOH or morphine Did not give high recovery with either protocol, and the recovery of 6-MAM was low in one protocol, but probably showed contamination results in the other.

C. Limits of Detection Table III shows the limits of detection for the class of drugs shown in Table II using the methods described in the listed examples. If more than one drug is listed, the limit of detection is for the drug with the highest level of contamination. The detection limit is NIDA (National Institute on Drug) for each of these drug classes.
Abuse) threshold level (if any) or below.

[0036]

[Table 3]

D. Comparison of Chromatographic Baselines Using the method described in the examples, a blank urine sample extract was loaded onto a high purity SDVB column and in each case using the extraction method provided by the manufacturer. Commercial PMMA
A clearer chromatographic baseline was obtained than the blank extract run over the column. Thus, by using this system, the likelihood of interfering with the analysis is reduced and the downtime of the instrument is reduced. This is illustrated in FIGS. 2-11, which show GC-MS scans of drug-loaded and blank samples for both high purity SDVB and PMMA columns, as summarized below. Figure Column Analyte Sample or Blank 2A SDVB THC-COOH Sample (30ng / ml) 2B Blank 3A PMMA THC-COOH Sample (30ng / ml) 3B Blank 4A SDVB Codeine Sample (Hydrolysate) 4B Morphine 300ng / ml 4C Codeine Blank (hydrolysate) 4D morphine 5A PMMA codeine sample (hydrolysate) 5B morphine 300ng / ml each 5C Codeine blank (hydrolysate) 5D morphine 6A SDVB codeine sample (non-hydrolysate) 6B standard 300ng / ml each 6C morphine 7A codeine blank (non-hydrolysate) 7B standard 7C morphine 8A PMMA codeine sample (non-hydrolysate) 8B standard 300ng / ml each 8C morphine 9A codeine blank (non-hydrolysate) 9B standard 9C morphine 10A SDVB Amphetamine sample 10B Amphetamine sample 300ng / ml each 10C methamphetamine 10D methamphetamine sample 11A amphetamine Min Blank 11B Amphetamine preparation 11C Metaamphetamine 11D Metaamphetamine preparation The extraction was performed according to the manufacturer's procedure for the PMMA column, and for the SDVB column, the procedure shown in the following example, namely, THC-COOH, Example 1 Opiates, Example 6E; and amphetamine / methamphetamine, performed according to Example 8.

In considering these figures, one must consider the difference in vertical scale shown in the upper left corner of each scan. For example, in Figures 2A-2B, the magnitude of the peaks in the lower figure is approximately three times larger (1.79% / 0.56%) than in the upper figure. Thus, the potential amount of THC-COOH shown in the blank sample that is likely to be disturbed is probably negligible relative to the sample peak when the drug is present at 30 ng / ml.

However, the corresponding pair of figures for the PMMA column (FIGS. 3A-3B, THC-COOH sample and blank) show signals that are more likely to interfere in blank samples (scale of sample scan). Less than twice). For the remaining scans, the blank extract against the extraction of opiate hydrolyzate shows potential interference for both columns (FIGS. 4C-D and 5C-D), but the peak of morphine interference from the PMMA column is SDVB Much more pronounced than for the column (FIGS. 5D and 4D, respectively). Blank scans for hydrolyzate extracts in SDVB had little or no interference (FIGS. 7A-7C), while PMM
For morphine in the A blank extract (FIG. 9), the interfering peak is clearly seen at approximately four times the scale of the sample extract (FIG. 8A). Finally, there is a potential for interference with methamphetamine and its deuterated preparation in the SDVB blank extract (Figures 11C-11D), but the scale of these scans is that of the corresponding sample (Figures 10C-D). It should be noted that it is several hundred times larger.

FIGS. 12A-12C show HPLC scans of (A) a TCA sample solution and (B) a urine sample with added drug (500 ng / ml each) and (C) a blank extract. Again, there is little or no disturbance in the blank baseline scan.

[0041]

【Example】

Hereinafter, the present invention will be described with reference to Examples, but it is not intended to limit the present invention. Purity underivatized SDVB resin 50mg to column materials and methods ^{9 × 1cm (Varian Bond Elut TM} ABS
Elut resin) and the height of the adsorbent bed was set to about 5 mm and used for extraction. The average particle size of this resin was about 125 μm, and the average pore size was about 450 °.

In each example, a known amount of an analyte was optionally added to the urine sample. Sample recovery in the eluate was quantified by comparison with a known amount of an internal standard added to the eluate before analysis. Unless otherwise noted, the buffer used was 0.1 M KH ₂ PO ₄ , pH 6. Ammonium hydroxide (NH ₄ OH) was a 30% aqueous solution stock solution. Unless otherwise noted, the GC-MS analysis used a 30 m × 250 μm DB5-ms column with the following temperature gradient: 100 ° C. to 280 ° C. at 10 ° C./min, then maintained at 280 ° C. for 12 minutes. Outline of solvent system 1. Washing: 2 ml of 5% acetonitrile aqueous solution containing 1% ammonium hydroxide Elution: ethyl acetate: isopropanol (85:15) 1 ml 2A. Washing: 0.1 ml KH ₂ PO ₄ (pH 6) 3 ml elution: acetonitrile : n-butyl chloride. (3: 7) 1ml 2B _{washing: 0.1M KH 2 PO 4 (pH} 6) 3ml elution: _{acetone: CHCl 3 (1: 1)} 2ml 3. washing: 5% aqueous methanol (95: 5 (MeOH: H ₂ O) 2 ml Elution: 2 times with 0.75 ml of methanol 4A. Washing: 0.1 M KH ₂ PO ₄ (pH 6) 1 ml Elution: hexane: ethyl acetate (3: 1) 2 ml 4B. Washing: 0.1 M KH ₂ PO ₄ (pH 6): acetonitrile (9: 1) 1 ml Elution: acetone: CHCl ₃ (1: 1) 2 ml 5 A. Washing: 0.1 M KH ₂ PO ₄ (pH 6): acetonitrile (9: 1) 1 ml elution : _{acetone: CH 2 Cl 2. (1} : 1) 2ml 5B _{washing: 0.1M KH 2 PO 4 (pH} 6): acetonitrile (9: 1) 1 ml elution: _{acetone: CHCl 3. (1: 1} ) 2ml 6A washed : 0.1 M KH ₂ PO ₄ (pH 6) 3 ml Elution: acetone: CHCl ₃ (7: 3) 2 ml 6 B. Washing: 0.1 M KH ₂ PO ₄ (pH 6) 3 ml Elution: meta Washing: 0.1 M KH ₂ PO ₄ (pH 6) 3 ml Elution: acetone: CHCl ₃ (1: 1) 2 ml 6D. Washing: 0.1 M KH ₂ PO ₄ (2% NH ₄ OH in MeOH (MeOH) 1.5 ml 6 C. pH 6) 3 ml Elution: acetone: 2% NH ₄ OH in CHCl ₃ (7: 3) 2 ml 6E. Adjust the pH of hydrolyzed urine to 6 Wash: 7% aqueous methanol (7:93 MeOH: H ₂ O) 2ml Elution: n-BuCl containing 1% NH ₄ OH: Ethyl acetate (1: 1) 1.5ml 6F. Adjust pH of hydrolyzed urine to 8.4 Wash / Elute: Same as 6F 7. Wash: 0.1M KH ₂ PO ₄ (pH 6) 1 ml Elution: acetone: CHCl ₃ (1: 1) 2 ml 8. Washing: 0.1 M KH ₂ PO ₄ (pH 6): acetonitrile (9: 1) 1 ml Elution: acetone: CHCl ₃ (1: 1) .) 2 ml 9A washing: 0.1M KH ₂ PO ₄ 3ml elution: CH ₂ Cl _2: isopropanol (80:20) 2 ml 9B washing:. 5% aqueous methanol _{(95: 5 MeOH: H 2} O) 3ml elution: CH ₂ Cl ₂ : isopropanol (80:20) 2ml 9C. Washing: 0.1M KH ₂ PO ₄ 3ml Elution: CH ₂ Cl ₂ : isopropanol: NH ₄ OH (78: 20: 2) 2ml 9D. Washing: 0.1M KH ₂ PO ₄ 3ml elution: methanol 2 ml 10. Washing: 3 ml of DI water or 3 ml of 5% methanol aqueous solution (95: 5 MeOH: H ₂ O) Elution: 2 ml of ethyl acetate Extraction method Example 1: Extraction method of THC-COOH 400 μl of 10 M KOH to 2 ml urine sample Was added. The solution was stirred and heated in a heating block at 60 ° C. for 20 minutes, then cooled to room temperature. 5% isopropanol (7
5%) and glacial acetic acid (25%) in a 0.2 M acetate buffer mixture at pH 4.0 (2 ml) were added, followed by thorough mixing.

The SDVB column was washed with 1 ml of methanol and the sample was added under gravity flow conditions. Then the column is 2m 5% acetonitrile aqueous solution containing 1% ammonium hydroxide
and dried by applying a vacuum adjusted to at least 7 "Hg for 5 minutes. The analyte was then eluted with 1 ml of ethyl acetate: isopropanol (85:15). (d ₃ -THC-COOH) was added to the eluate and the solvent was removed at room temperature under nitrogen.
To this residue was added 50 μl of BSTFA (N, O-bis (trimethylsilyl) trifluoroacetamide) containing 1% TMCS (trimethylchlorosilane), followed by stirring. The mixture was incubated at 70 ° C. for 20 minutes and cooled to room temperature. Then GC the sample
Injected into -MS column. Example 2A: Method for extracting benzoylecgonine and cocaine Mix 2 ml of urine and 2 ml of buffer in a large test tube and add phosphoric acid if necessary
The pH was adjusted between 5.0 and 7.0.

The column was washed sequentially with 2 ml of methanol and 2 ml of buffer. The sample was poured into the column reservoir and allowed to slowly flow through the column under light vacuum. The column was then washed with 3 ml of buffer and dried under full vacuum for 5 minutes.
The analyte was eluted with 1 ml of acetonitrile: n-butyl chloride (3: 7). An internal standard (nalorphine) was added to the eluate and then evaporated to dryness under a gentle stream of nitrogen. The analyte was treated with 50 μl of BSTFA containing 1% TMCS and derivatized by heating at 70 ° C. for 20 minutes. Aliquots of 1-2 μl were used for GC-MS analysis. Example 2B: Alternative Method for Extraction of Benzoyl Ergonine and Cocaine Acetonitrile: Acetone: CHCl ₃ (1: 1) instead of n-butyl chloride as elution solvent
The procedure of Example 2A was followed except that 2 ml was used. Example 3: Extraction method of amphetamine and methamphetamine 2 ml urine sample was diluted with 1.5 ml buffer. The column was washed with 2 ml of methanol followed by 2 ml of buffer. The sample was applied under gravity flow conditions, and then the column was washed with 2 ml of 5% aqueous methanol. The column was dried under reduced pressure of 5 inches Hg or more, and the analyte was eluted using two aliquots of 0.75 ml of methanol.

An internal standard (d ₅ -amphetamine and d ₅ -methamphetamine) was added to the eluate,
Then, 30 μl of methanolic hydrochloric acid (methanol: concentrated hydrochloric acid, 9: 1) was added. The eluate was evaporated to dryness under nitrogen, 50 μl of pentafluoropropionic anhydride (PFPA) was added and the solution was heated at 70 ° C. for 40 minutes. Aliquot 1 μl GC-M with the following temperature gradient
Used for S analysis: 80 ° C. to 240 ° C. at 10 ° C./min, then maintained at 240 ° C. for 4 minutes. Example 4A: Extraction method of barbiturates In a large test tube, 2 ml of urine sample was diluted with 2 ml of buffer.

The column was washed with 1 ml of MeOH and then with 1 ml of buffer and the sample was applied under gravity flow conditions. The column was then washed with 1 ml of buffer and dried by applying reduced pressure adjusted to at least 7 ¹¹ Hg for 5 minutes. The analyte was eluted with 2 ml of hexane: ethyl acetate (3: 1). The eluate was transferred to Reacti-Vial ^™ and evaporated to dryness at room temperature under nitrogen. The solution - was stirred after addition of (d ₅ secobarbital). Then sample G
Injected into a C-MS column. Example 4B: Alternative method of barbiturates extraction method Buffer / acetonitrile (9: 1) 1 ml as washing solution and acetone: CHC
The procedure described in Example 4A was followed except that 2 ml of l ₃ (1: 1) was used for elution. Example 5A: Method of extracting PCP In a large test tube, 2 ml of urine and 2 ml of buffer were mixed and then, if necessary, the pH was adjusted between 5.0 and 7.0 by adding phosphoric acid.

The column was washed sequentially with 2 ml of methanol and 2 ml of buffer. The sample was poured into the column reservoir and allowed to slowly flow through the column under light vacuum. The column was then washed with 1 ml of buffer: acetonitrile (9: 1) and dried under full vacuum for 5 minutes. The analyte was eluted with 2 ml of acetone: CH ₂ Cl ₂ (1: 1). An internal standard (carbazole) was added to the eluate, then evaporated to dryness under a gentle stream of nitrogen and reconstituted in 100 μl of ethyl acetate. Aliquot 1-2 μl of this solution
Used for GC-MS. Example 5B: Alternative method of PCP extraction method The procedure described in Example 5B was followed except that acetone: CHCl ₃ (1: 1) 2 ml was used instead of acetone: CH ₂ Cl ₂ as the elution solvent. Example 6A: Opium (Non-Hydrolysate) Extraction Method 1.5 ml of buffer was added to a 2 ml urine sample. The column was washed with 2 ml of methanol and then with 1.5 ml of buffer. The sample was applied under gravity flow conditions, then the column was washed with 3 ml of buffer. The column was dried under reduced pressure of 7 inches Hg or more for 5 minutes, then the drug was eluted with 2 ml of acetone: CHCl ₃ (7: 3).

The eluate is recovered in or transferred into Reacti-Vial ^™ and
The deuterated internal standards like d ₃ was added into a vial. The contents were evaporated to dryness under nitrogen. BSTFA or MSTFA containing 50 μl of acetonitrile and 1% TMCS (
(N-methyl-N-trimethylsilyl trifluoroacetamide) (50 μl) was added, and the content was heated at 70 ° C. for 30 minutes. An aliquot of 1 μl was injected onto the GC column with the following temperature gradient: 3.1 min at 80 ° C., increasing at 20 ° C./min to 285 ° C., then maintaining at 285 ° C. for 5.85 min. Example 6B-D: Alternative Method for Extracting Opium (Non-Hydrolysate) The procedure described in Example 2A was followed except that the following solvents were used for elution in place of acetonitrile: n-butyl chloride. 6B: 1.5 ml of methanol (MeOH) containing 2% NH ₄ OH 6C: 2 ml of acetone: CHCl ₃ (1: 1) 6D: 2 ml of acetone: CHCl ₃ (7: 3) containing 2% NH ₄ OH 6E: Extraction method of opium (hydrolyzate) 0.25 ml of concentrated hydrochloric acid was added to 1 ml of urine sample. The sample was heated at 120 ° C. for 30 minutes and cooled to room temperature. To this solution was added 0.35 ml of 10 M KOH followed by 1.5 ml of buffer.

The column was washed with 1 ml of methanol followed by 0.5 ml of buffer. The sample was applied under gravity flow conditions, then the column was washed with 2 ml of 7% aqueous methanol. The column was dried under reduced pressure of 7 inches Hg or more for 5 minutes, then the drug was eluted with 1.5 ml of ethyl acetate: n-butyl chloride (1: 1) containing 1% ammonium hydroxide. The eluate was collected and processed and analyzed as described in Example 6A above.

The hydrolysis process was omitted, and this method was found to be effective also for free opium codeine (90% recovery) and 6-MAM (89% recovery). Example 6F: Alternative Method for Extracting Opium (Hydrolysate) The procedure described in Example 6E was followed except that the pH of the hydrolyzed urine was adjusted to 8.4 before application to the column. Example 7: Extraction method of benzodiazepines Urine sample preparation and column conditioning were performed as described in Example 2A,
The sample was applied to the column. The column was then washed with 2 ml of buffer and dried for 5 minutes under full vacuum. The analyte was eluted with 2 ml of acetone: CHCl ₃ (1: 1).

An internal standard (nalorphine) was added to the eluate and then evaporated to dryness under a gentle stream of nitrogen. Analytes were derivatized by treatment with 50 μl BSTFA + 1% TMCS and heating at 70 ° C. for 20 minutes. Aliquots of 1-2 μl were used for GC-MS analysis. Example 8: LSD extraction method Urine sample preparation and column conditioning were performed as described in Example 2A, and the sample was applied to the column. Then 1 ml of buffer: acetonitrile (9: 1)
And dried under full vacuum for 5 minutes. Acetone: CHCl ₃ (1: 1) 2
The analyte was eluted using ml. Adding an internal standard (d ₃ -LSD) in the eluate, then dried under a gentle stream of nitrogen was derivatized analytes as described in the previous examples 7.

The method also includes THC-COOH (91%), cocaine / benzoylecgonine (100%), barbiturates (87-100%), PCP (100%), and opiates (75-85%) Also showed good recovery. Example 9A: Tricyclic antidepressant extraction method Urine sample preparation, column conditioning as described in Example 2A,
The sample was applied to the column. The column was then washed with 2 ml of buffer and dried under reduced pressure of 5 inches Hg or more for 5 minutes. The analyte was eluted with ₂ ml of CH ₂ Cl ₂ : isopropanol (80:20).

[0053] An internal standard (trimipramine) was added to the eluate and then dried under nitrogen. The dried eluate was redissolved in 70 μl of methanol and 130 μl of the following mobile phase B (0.01 M KH ₂ PO ₄ ). A sample of 90 μl was analyzed by HPLC using diode array detection (DAD) as follows: Column: Varian TCA, 4.6 mm × 15 cm Mobile phase: A: 80:20 acetonitrile: 0.01 M KH ₂ PO ₄ (pH 6) B: 0.01 M KH ₂ PO ₄ (pH6) Gradient: isocratic -38% A: 62% B Flow rate: 1.3 ml / min Detection: UV at 220 nm Example 9B-D: Alternative method of extracting tricyclic antidepressants except for substitution according to the method of example 9A according: 9B cleaning:.. 5% aqueous methanol 3 ml 9C elution: CH ₂ Cl _{2: isopropanol: NH 4 OH. (78:} 20: 2) 2ml 9D elution: methanol 2 ml Example 10: General Extraction / Screening Method for Drugs of Abuse This method involves the use of THC-COOH, opium, amphetamines, etc. as shown in Table II above.
It provides good recovery for a wide range of drugs and / or metabolites, including barbiturates, PCP, LSD, cocaine and benzodiazepines.

In a large test tube, mix 2 ml urine with 2 ml buffer and add phosphoric acid if needed
The pH was adjusted between 5.0 and 7.0. The column was washed sequentially with 2 ml of methanol and 2 ml of buffer. The sample was poured into the column reservoir and allowed to slowly flow through the column under light vacuum. The column is charged with 3 ml of DI water or, alternatively, 3 ml of 5% aqueous methanol (5:95
MeOH: washed with H ₂ O), dried for 5 minutes under full vacuum. The analyte was then eluted with 2 ml of ethyl acetate. An internal standard and / or a derivatizing reagent was added to the eluate, and the analyte was quantified as described in Example 1-9 above.

Although the invention has been described with respect to particular methods and examples, it will be understood that various modifications, particularly with respect to dilution volumes, conditioning, washing and elution solvents, may be made within the scope of the invention.

[Brief description of the drawings]

FIG. 1 shows a preferred configuration of an extraction column used to carry out the extraction method of the present invention.

FIGS. 2A-2B show (A) SDVB extract prepared according to the invention of a urine sample supplemented with 15 ng / ml of THC-COOH, and (B) a similar extract of a blank sample without drug. 3 shows a GC-MS scan of the sample.

FIG. 3A-3B shows a GC-COOH sample extract and blank extract similar to FIG. 2A-2B prepared on a commercially available PMMA (polymethyl methacrylate) extraction column according to the manufacturer's instructions. Show MS scan.

FIG. 4A-4D shows the (A, B) NIV detection limit level (300 ng / ml), the SDVB extract of the hydrolyzed urea sample to which each of these drugs was added, and the addition of the (C, D) drug. (A, C) codeine ion region and (B, D) of GC-MS scan of similar extract of blank sample
) Shows the morphine ion region.

FIGS. 5A-5D are GCs of (A, B) hydrolyzed codeine / morphine samples and PMMA extracts of (C, D) blank extracts prepared according to the manufacturer's instructions, as in FIGS. 4A-4D. -Indicates the corresponding area of MS scan.

FIGS. 6A-6C show codeine, internal standard, and morphine of GC-MS scans of SDVB extracts of non-hydrolyzed urine samples to which each of these drugs was added at the detection limit level of NIDA (300 ng / ml). Each shows an ion region.

FIGS. 7A-7C show GC-GCs of SDVB extracts of blank non-hydrolyzed urine samples without added drug.
The corresponding area of the MS scan is shown.

FIG. 8A-8C shows the corresponding area of a GC-MS scan of a PMMA extract of a non-hydrolyzed urine sample supplemented with codeine and morphine as in FIGS. 6A-6C, prepared according to the manufacturer's instructions. Show.

FIGS. 9A-9C show the corresponding areas of a GC-MS scan of a PMMA extract of a blank non-hydrolyzed urine sample with no drug added, prepared according to the manufacturer's instructions.

FIGS. 10A-10D show amphetamine (B), methamphetamine (D) and GC-MS scans of a SDVB extract of a urine sample to which each of these drugs was added at the detection limit level of NIDA (300 ng / ml). The internal standard (A, C) ion region is shown.

FIGS. 11A-11D show corresponding areas of a GC-MS scan of an SDVB extract of a blank urine sample with no added drug.

12A-12C show (A) a standard TCA drug mixture, (B) an SDVB extract of a urine sample to which each of these drugs was added at 500 ng / ml, and (C) an SDVB of a blank urine sample to which no drug was added. 2 shows an HPLC trace of the extract.

Claims (27)

[Claims] An aqueous bodily fluid sample suspected of containing an abused drug or an analyte that is a metabolite or derivative thereof is contacted with a substantially underivatized styrene-divinylbenzene resin adsorbent, whereby Allowing the analyte to adsorb to the adsorbent; washing the adsorbent with a first solvent system effective to selectively remove non-analyte components from the adsorbent while the analyte remains bound to the adsorbent; Applying to the sorbent a second solvent system effective to selectively elute the analyte from the sorbent; the solvent system passing through the sorbent by gravity flow or by applying a reduced pressure; Yields an eluate containing at least about 80% of the analyte present in the sample, which is suitable for gas chromatography analysis of the analyte without further purification; further included in the eluate Analyte detection or quantification diagnose how. 2. The method of claim 1, wherein said eluate contains about 90% or more of the analyte present in said sample. 3. The abuse drug or metabolite is THC carboxylic acid, heroin, morphine, 6-acetyl morphine, morphine-3-glucuronide, codeine, cocaine, benzoylecgonine, ecgonine methyl ester, amphetamine, methamphetamine,
Lysergic acid diethylamide (LSD), phencyclidine (PCP), barbiturate,
2. The method of claim 1, wherein the method is selected from the group consisting of benzodiazepines or metabolites thereof, and tricyclic antidepressants or metabolites thereof. 4. The method according to claim 3, wherein the barbiturate is selected from the group consisting of butabarbital, pentobarbital, secobarbital, phenobarbital, alfenal and amobarbital. 5. The method according to claim 3, wherein the benzodiazepine is oxyazepam or chlorodiazepoxide, and the metabolite is hydroxyalprazolam or hydroxytriazolam. 6. The method of claim 3, wherein the tricyclic antidepressant is doxepin, imipramine, or amitriptyline, and the metabolite is nordoxepin, desipramine, or nortriptyline. 7. The styrene-divinylbenzene adsorbent comprises 10 ml of hexane and then 1 ml of methanol.
2. The method according to claim 1, wherein the resin is a high-purity resin such that extraction of about 1.0 g of resin with 0 ml results in less than about 0.2 mg of residue. 8. The method according to claim 1, wherein the detection or quantification is performed by gas chromatography (GC), GC-mass spectrometry (MS),
MS-MS or High Performance Liquid Chromatography (HPLC: High Performance Liquid Chro
The method according to claim 1, which is performed by matography). 9. The method of claim 1, wherein said aqueous body fluid sample is a urine sample. 10. The method of claim 2, wherein the application, washing and elution steps on 2 ml of the urine sample comprise about 100
10. The method according to claim 9, wherein less than mg of resin is used. 11. The method according to claim 1, wherein the first solvent system is water and the second solvent system is ethyl acetate.
Method according to 1. 12. The method according to claim 1, wherein said first solvent system is 5% aqueous methanol and said second solvent system is ethyl acetate. 13. The first solvent system is a weakly acidic aqueous buffer containing about 10% by volume acetonitrile, and the second solvent system is acetone / chloroform at a volume ratio of about 1: 1. The method of claim 1, wherein 14. The method according to claim 1, wherein the analyte is THC-COOH and the first solvent system is aqueous acetonitrile containing about 5% by volume of acetonitrile and 1% by volume of a 30% aqueous ammonium hydroxide solution in water. The method of claim 1, wherein the second solvent system is ethyl acetate / isopropyl alcohol in a volume ratio of about 85:15. 15. The method according to claim 15, wherein the analyte is opium selected from the group consisting of heroin, morphine, 6-acetylmorphine, and codeine, or a metabolite or derivative thereof,
2. The method of claim 1, wherein the solvent system is a weakly acidic aqueous buffer and the second solvent is methanol containing about 2% by volume of a 30% aqueous ammonium hydroxide solution. 16. The method according to claim 16, wherein the analyte is opium selected from the group consisting of heroin, morphine, 6-acetylmorphine, morphine-3-glucuronide and codeine, or a metabolite or derivative thereof, Prior to hydrolysis, wherein the first solvent system is aqueous methanol containing about 7% methanol by volume in water;
The method of claim 1, wherein the second solvent system is n-butyl chloride / ethyl acetate in a volume ratio of about 1: 1 containing about 1% by volume of a 30% ammonium hydroxide solution. 17. The method of claim 17, wherein the analyte is cocaine or benzoylecgonine, the first solvent system is a weakly acidic aqueous buffer, and the second solvent system is acetone / chloroform in a volume ratio of about 1: 1; Or the method of claim 1, wherein the ratio is acetonitrile / n-butyl chloride in a volume ratio of about 3: 7. 18. The method according to claim 18, wherein the analyte is amphetamine or methamphetamine, the first solvent system is aqueous methanol containing about 5% by volume methanol in water, and the second solvent system is aqueous methanol.
2. The method of claim 1, wherein said solvent system is methanol. 19. The method according to claim 19, wherein the analyte is butabarbital, pentobarbital, secobarbital,
A barbiturate selected from the group consisting of phenobarbital and amobarbital, wherein the first solvent system is a weakly acidic aqueous buffer, and the second solvent system is about 3: 1 by volume hexane / ethyl acetate; Or the method of claim 1, wherein the volume ratio is acetone / chloroform of about 1: 1. 20. The analyte is PCP, the first solvent system is a weakly acidic aqueous buffer, and the second solvent system is about 1: 1 acetone / chloroform or acetone / methylene chloride in a volume ratio of about 1: 1. The method of claim 1, wherein 21. The method according to claim 21, wherein the analyte is selected from benzodiazepine, oxazepam, chlorodiazepoxide, hydroxyalprazolam and hydroxytriazolam.
2. The method of claim 1, wherein said solvent system is a weakly acidic aqueous buffer and said second solvent system is acetone / chloroform in a volume ratio of about 1: 1. 22. The method according to claim 19, wherein the analyte is LSD, the first solvent system is a weakly acidic aqueous buffer containing about 10% by volume acetonitrile, and the second solvent system is about 1: 1 by volume. 2. The method according to claim 1, which is acetone / chloroform. 23. The method according to claim 23, wherein the analyte is a tricyclic antidepressant selected from the group consisting of doxepin, imipramine, amitriptyline, nordexepin, desipramine and nortriptyline, or a metabolite thereof, and the first solvent system is weakly acidic. Aqueous buffer or
The method of claim 1 wherein the second solvent system is 5% aqueous methanol and the second solvent system is methylene chloride / isopropyl alcohol in a volume ratio of about 4: 1. 24. The method of claim 23, wherein said first solvent system is a weakly acidic aqueous buffer. 25. A method for increasing the flow of liquid through a column having an adsorbent packed bed and having a discharge tube connected to the underside of the bed, the method comprising providing a tapered tip at the lower end of the discharge tube, thereby providing a capillary. A method comprising facilitating the flow of an adsorbent bed and a drain through action. 26. The method according to claim 25, wherein said solvent is an aqueous solvent or an aqueous solution. 27. The method of claim 25, wherein said sorbent is a silica-based sorbent.